Our work on the regulation of immunoglobulin kappa gene expression has further defined the functional elements of the enhancer located downstream of the kappa constant region gene, which is a candidate target for gamma- interferon regulation of kappa expression. We constructed plasmids containing this region of DNA linked to a reporter gene, and transfected these plasmids into the human B lymphoid cell CA46. By examining which sequences led to gene expression, we could define a segment of about 170 bp that contained most of the enhancer activity. This segment was examined for evidence of binding of proteins in vivo by a DNA footprinting technique involving ligation-mediated polymerase chain reaction; the technique demonstrated four motifs with alterations in reactivity to dimethylsulfate suggestive of B cell specific DNA-protein interactions. These four motifs include two that were found to be important for function of the homologous murine enhancer, as well as two that, while well- conserved in the murine sequence, were not found to be functional. We have examined these four regions for enhancer activity and for the ability to bind B cell nuclear proteins in vitro. Enhancer function was assessed first by a series of constructs with small deletions, and more recently by an extensive series of mutation constructs; these experiments have documented the enhancer activity of three of the motifs and have begun to characterize sequence requirements for each one. In vitro protein binding to these motifs has been assessed by gel retardation experiments. Sequence-specific binding to synthetic oligonucleotides including each of the three functional motifs has been demonstrated in B cell nuclear extracts. We are currently comparing the sequence requirements for protein binding and enhancer activity and exploring the distribution of the binding proteins in lymphoid and non-lymphoid cells.